Biomarker Research

Improving Proteomics Reproducibility using Chip-Based Nanoflow Chromatography:  Two-Column Switching Workflows Enabled by the Eksigent cHiPLC®-Nanoflex with the NanoLC-Ultra® System

Biomarker discovery using NanoLC/MS typically requires the comparison of a control sample with a diseased or treated sample to elucidate differences and ultimately identify candidate biomarkers. Once putative biomarkers are discovered they are often then verified by rigorously analyzing multple samples for detection and confirmation of the candidate biomarkers of interest. For both discovery and verification, the requirements for highly reproducible retention times are tantamount to success.  However, historically work utilizing NanoLC gradients required a flow splitter prior to the chromatographic column to achieve NanoLC flow rates. This approach is not always able to provide a constant flow to the NanoLC column due to backpressure differences between the NanoLC column and the splitter and results in a systematic drift in retention time. Additionally, making low dead-volume connections is a technique-dependent art for many users. And finally the low solvent flow rate means that loading sample, running gradients, and re-requilibrating columns takes a long time, limiting throughput.

The Eksigent cHiPLC®-nanoflex is a microchip platform that allows columns and traps to be easily installed for plug-and-play simplicity, without any sacrifice in chromatographic fidelity. This flexible system allows for direct injection, trap-elute, two column switching and serial two-column NanoLC/MS workflows. Microfabrication technology for producing the microfluidic chips ensures excellent column-to-column reproducibility (<2%) and the unique connection system makes changing columns and traps easy with precise low dead-volume connections made easily every time. Coupled with the splitless Eksigent nanoLC Ultra® system, the stability of the system enables very long batches. Additionally, the system enables a multiplexed two-column switching workflow that maximizes MS duty cycle by putting autosampler, washing and regeneration steps offline. The throughput enabled by this efficient workflow means the MS time is approaching 100% utilization and many more samples can be run per day.

Read the Application Note in its entirety.